Summary: The aims of this research project are to understand the regulation of viral gene expression during herpes simplex virus (HSV) infection, evaluate the feasibility of herpesvirus vectors as gene delivery vectors, and determine the herpesvirus genes that would be most effective for development as potential subunit vaccines. Understanding the regulation of HSV gene expression is essential for the development and evaluation of successful vaccines, particularly live virus vaccines. Similarly, development and evaluation of safe and effective HSV vectors, and the regulated expression of foreign genes in such vectors, requires an understanding of the mechanisms that regulate gene expression. One area of our research is designed to investigate methods which might allow regulated expression of foreign genes in these vectors. In FY01, novel replication- incompetent herpes simplex viruses have been constructed which express both a tetracycline- transactivator and a lacZ marker gene, each of which is under the control of a tetracycline-response element. This permits induction of the lacZ gene by the antibiotic tetracycline in any cell type that can be infected with the vector. We have shown that gene expression from such vectors can be regulated by tetracycline addition and can persist for extended periods of time in post-mitiotic neurons. Vectors of this type may be especially useful for regulated gene delivery to neurons in vivo and in vitro. In addition, we have cloned a replication- defective HSV vector as a bacterial artificial chromosome. This system should facilitate construction of vectors by permiting genetic manipulation of the vector in a prokaryotic host. Another part of this research project is designed to evaluate various HSV gene products as potential components of subunit vaccines, and to evaluate different delivery strategies in order to maximize a protective immune response. Safe and effective vaccines for genital herpes have proven difficult to develop and will likely require a greater understanding of the interaction of the virus with its host as well as the requirements for generation of protective immunity. In FY01, we have evaluated a prime-boost strategy for enhancing the protective immune response to HSV2 antigens. We have used a plasmid DNA vector that expresses the HSV2 glycoprotein D gene in combination with a Modified Vaccinia Virus Ankara (MVA) vector expressing the same gene. Boosting plasmid delivered gD with MVA-gD substantially increases the antibody response as well as the cellular immune response, compared to boosting with gD-DNA. Interestingly, priming with MVA-gD followed by gD-DNA appears to elicit the broadest and most vigorous humoral and cellular response to gD. Work is underway to further evaluate this and other vaccination regimens for their ability to enhance protective immune responses to HSV2.